Leukemia (2009) 23, 829–833 & 2009 Macmillan Publishers Limited All rights reserved 0887-6924/09 $32.00 www.nature.com/leu EDITORIAL The rewards and challenges of array-based karyotyping for clinical oncology applications Leukemia (2009) 23, 829–833; doi:10.1038/leu.2009.24 the arrays used for karyotyping include SOMA (SNP oligonu- cleotide microarrays)7 and CMA (chromosome microarray).8,9 Some consider all platforms to be a type of array comparative Array-based karyotyping is a powerful new technique for genomic hybridization (arrayCGH), whereas others reserve that assessing chromosomal copy number changes that provides term for two-dye methods, and still others who segregate SNP information not previously obtainable by fluorescent in situ arrays because they generate more and different information hybridization (FISH) or conventional cytogeneticsFwhich can than two-dye arrayCGH methods. be both a blessing and a challenge. In this issue, Gunn et al.1 Regardless of the name of the assay or the probe types used, present atypical 11q deletions identified by array-based array-based karyotyping is becoming standard of care for many karyotyping of chronic lymphocytic leukemia (CLL) that may genetic applications and is now on the verge of bursting into be missed by FISH panels used for prognostic stratification of clinical oncology. CLL is an ideal neoplasm to study with copy this disease. Array-based karyotyping is gaining acceptance as a number arrays because (1) the genetic lesions with known clinical tool, and physicians should be prepared to judiciously clinical relevance are chromosomal gains and losses rather interpret results from these platforms. The advantages of array- than balanced translocations and inversions, (2) DNA from a based karyotyping are many and vary somewhat depending on fresh sample is generally available making the analysis more what kind of array is used, but they include high-resolution, straightforward than that for DNA obtained from formalin-fixed genome-wide copy number assessment in one assay; a paraffin-embedded tissue, (3) the tumor burden is known from permanent, numeric result that does not fade over time like the flow cytometry results and can help guide downstream the fluorescent signals of FISH; the ability to karyotype formalin- analysis, (4) the tumor burden tends to be relatively high in the fixed paraffin-embedded tissues and the simultaneous capture of peripheral blood and (5) enrichment for B cells or CLL cells is loss-of-heterozygosity (LOH) status if using a single-nucleotide simple, cost effective and amenable to routine clinical use, polymorphism (SNP)-based array. However, because arrays can which minimizes the effect of ‘normal clone contamination.’ display the genome at high resolution, it is becoming apparent Several groups have recently published manuscripts using copy that the individual molecular lesions identified earlier by FISH number or SNP arrays to study CLL or to validate them for are actually heterogeneous in both genomic length and copy clinical use,5,10–13 and the technique has been successfully number. The clinical meaning of these different subtypes of applied to several solid2–4,14,15 and liquid16–19 tumors. genetic lesions, if any, is yet to be determined. In addition, many In CLL, it is typical to perform a standard FISH panel to genomic changes of uncertain clinical significance are identified determine copy number at key regions of the genome with by these platforms, and there are no standards for reporting such well-established clinical significance, including 6q, 11q, chro- lesions or for archiving them, so that the reports can be amended mosome 12, 13q14 and 17p. In this issue, Gunn et al.1 use a as our knowledge of these lesions evolve. clinically validated array customized to interrogate all known Array-based karyotyping can be carried out with several CLL prognostic loci (179 probes) and 914 FISH-mapped linearly different platforms, both laboratory developed and commercial. distributed clones for whole-genome coverage at an average The arrays themselves can be genome-wide with probes resolution of approximately 2.5 Mb. Their manuscript highlights distributed over the entire genome, or targeted with probes for four atypical 11q deletions. These 11q lesions were considered genomic regions known to be involved in a specific disease or atypical because they did not include the ATM gene, and two of group of diseases, or a combination of both. Furthermore, array- the deletions were missed by the commercial FISH probe used based karyotyping can be carried out with ‘copy number only’ for this locus (11q22.3 Vysis LSI ATM probe, Abbott Molecular, arrays or SNP arrays, which can provide both copy number and Des Plaines, IL, USA). The loss of the ATM tumor suppressor LOH status. The probe types used for ‘copy number only’ arrays gene (TSG), located at 11q22, is often implicated in the include cDNA, BAC clones and oligonucleotides (for example, pathogenesis of CLL. However, it is unlikely to be the sole Agilent (Santa Clara, CA, USA), Nimblegen (Madison, WI, cause of the 11q abnormality, as the minimally deleted region of USA). Commercially available SNP arrays can be solid phase 11q houses other potential candidate TSGs, such as RDX and (Affymetrix, Santa Clara, CA, USA) or bead-based (Illumina, San FDX1 genes,20 and not all CLL patients with deletions of 11q Diego, CA, USA). Some arrays may contain both polymorphic have evidence of an ATM mutation of the remaining allele.21 (SNP-containing) and non-polymorphic (copy number only) Other groups have reported data suggesting that there is a probes, such as the Affymetrix SNP 6.0 array. The actual slightly more telomeric but overlapping region that does not resolution of the virtual karyotype will depend primarily on the include ATM.21–23 This finding was corroborated by Lehmann probe density, the probe performance, the quality of the DNA et al.5 who also observed a second commonly deleted region at and the analysis software. Despite the diversity of platforms, 11q that is telomeric to the ATM gene when they used SNP ultimately they all use genomic DNA from disrupted cells to arrays to karyotype CLL samples. It is postulated that this region recreate a high-resolution karyotype in silico. The end product may harbor another TSG associated with the development of does not yet have a consistent name and has been called virtual CLL, and that concurrent deletion of ATM and this other TSG karyotyping,2,3 digital karyotyping,4 molecular allelokaryotyp- may contribute to a poor prognosis. It is therefore likely that a ing5 and molecular karyotyping.6 Other terms used to describe single FISH probe cannot capture all clinically relevant lesions Editorial 830 at this locus. This article underscores how copy number arrays not only allow us to detect lesions missed by FISH, but also enable us to further refine the break points of 11q deletions and help to determine the clinical relevance, if any, of the subtypes of 11q lesions. Aside from variation in the genomic length of the 11q deletions described by Gunn et al.,1 other types of genetic heterogeneity at clinically relevant loci in CLL have been elucidated by array- based karyotyping. Sargent et al.10 report clinical validation data for a custom CLL oligonucleotide array and highlights length heterogeneity at the 13q14 locus. Patel et al.24 report their clinical validation of a custom CLL BAC array and also underscore the length heterogeneity seen at the 13q14 locus. Ouillette et al.25 used 50 K Affymetrix SNP arrays to subtype 13q14 lesions based on copy number (bi- or mono-alleleic loss) and break points, and they propose a subclassification of 13q14 lesions based on length and gene dose heterogeneity of this locus. Although the lesions detected by FISH have generally been thought of as monolithic, most physicians can readily con- ceptualize length heterogeneity of these deletions. However, Figure 1 Cellular basis of regional heterogeneity seen with array- karyotyping with high resolution using arrays is bringing less based karyotyping. Heterozygous deletions are light blue and easily conceptualized genetic lesions into play, such as copy homozygous deletions are dark blue as depicted within a chromosome neutral LOH (acquired uniparental disomy (UPD)) and regional pair in each cell. (a) Clonal evolution, (b) two separate clones, both scenarios will result in apparent regional copy number heterogeneity copy number heterogeneity. UPD refers to a chromosomal when subjected to array-based karyotyping. region in which both copies of that region are acquired from the same parent, resulting in a copy number of two but with LOH (thus, copy neutral LOH). This can occur through mitotic recombination, chromosome non-dysjunction and loss of one parental chromosome with duplication of the other. Copy neutral LOH can act as the ‘second hit’ of the Knudson two hit hypothesis of tumorigenesis, similar to a deletion, resulting in the removal of the remaining wild-type allele of a TSG.26 Copy neutral LOH is reported to constitute 20–80% of the LOH seen in human cancers, both solid and liquid.16,27–30 Using Affymetrix SNP arrays, Pfeifer et al.12 and Lehmann et al.5 identified copy neutral LOH at clinically relevant loci in CLL. This is noteworthy because conventional cytogenetics, FISH or ‘copy number only’ arrays cannot detect this type of lesion. Array-based karyotyping can also readily detect heterogeneity of copy number state and gene dosage within a particular chromosomal regionFso called ‘regional copy number hetero- geneity.’ For example, a region of 13q14 can be deleted in both chromosomes (homozygous deletion), whereas an adjacent, intervening or overlapping genomic region is deleted in only Figure 2 13q14 regional copy number heterogeneity and efficacy of one chromosome (heterozygous deletion). Figure 1a depicts B-cell enrichment. (a) Single-nucleotide polymorphism (SNP) array- clonal evolution of a disease-associated locus, such as 13q14, based karyotype of the 13q14 locus of a chronic lymphocytic showing a single chromosome pair in each cell.